DE69833031T2 - ALKALINE PROTEASE - Google Patents

Description

Technical area

The The present invention relates to an alkaline protease known as a detergent incorporated in a detergent is useful to encode a same Gene, a microorganism producing the same and a detergent composition, which contains the same.

Background of the invention

protease has been used in a variety of detergents, such as laundry detergents, cosmetic Compositions, bath additives, Food modifying and pharmaceutically effective Means like digestive aids and anti-inflammatory drugs widely used.

From these are the proteases used in detergents in the largest amounts on an industrial scale produced and therefore make up a significant part of the merchandise out. examples for such proteases include Alcalase, Savinase (product of Novo Nordisk), Maxacal (product of Genencor), Blap (product of Henkel) and protease K (KAP, product of Kao Corporation).

meanwhile Attempts have been made to use the performance of detergents To improve enzymes. For example, a disclosure discloses Japanese Patent Application (kokai) No. 6-70765 discloses an enzyme having a high stability across from Heat and a surface-active Substance. Japanese Patent Application Laid-Open (kokai) No. 9-121855 discloses an enzyme which refers to insoluble proteins such as keratin acts and has a high specific activity. The Japanese revealed Patent Applications (kokai) Nos. 5-211868 and 9-121856 disclose Enzyme with excellent activity in a low temperature range. The European Patent No. 0130756 discloses a method for increasing the Stability of a Enzyme opposite an oxidizing agent.

In many cases Contains dirt on laundry a variety of components such as lipids and solid particles that are not protein. Therefore, there is a need for a detergent having an excellent Washing power opposite such complex types of dirt. To meet the requirement, In general, a variety of enzymes and surfactants have been identified Substances incorporated in a detergent.

Also When a variety of enzymes are incorporated, theirs can Effects, however, are not fully exercised when the enzymes unstable in the presence of complex soils and none show constant and sufficient activity. Conventional enzymes are not satisfactory in this regard.

Disclosure of the invention

Regarding from the above, the present inventors have an alkaline one Protease discovers which even in the presence of a fatty acid in one high concentration has a constant casein-degrading activity and even under complex soil conditions, eg. B. types of dirt, which Containing protein and sebum, showing excellent detergency.

• (a) eine Aminosäuresequenz, wie in SEQ ID NO: 1 oder 2 gezeigt, umfasst oder
• (b) welche die Aminosäuresequenz von (a) umfasst, in welcher eine Sequenz, in der eine oder mehrere Aminosäuren deletiert, substituiert oder hinzugefügt sind, die folgenden physico-chemischen Eigenschaften hat:
• (i) Wirkungs-pH-Bereich über einen breiten pH-Bereich von 4–13 wirkend und bei einem pH-Wert von 6–12 80% oder mehr der Aktivität beim pH-Optimum zeigend;
• (ii) Stabiler pH-Bereich über einen pH-Bereich von 6–11 stabil, wenn bei 40°C für 30 Minuten behandelt;
• (iii) Isoelektrischer Punkt mit einem isoelektrischen Punkt von etwa 8,9–9,1 und
• (iv) Wirkung einer Fettsäure eine Casein-abbauende Aktivität, die nicht durch Oleinsäure gehemmt wird,

worin die Aminosäure zu 90% oder mehr zu SEQ ID NO: 1 oder 2 identisch ist.Accordingly, in one aspect of the present invention, there is provided an alkaline protease which:

• (a) comprises an amino acid sequence as shown in SEQ ID NO: 1 or 2, or
• (b) which comprises the amino acid sequence of (a) in which a sequence in which one or more amino acids are deleted, substituted or added has the following physicochemical properties:
• (i) acting pH range over a broad pH range of 4-13 and exhibiting at pH 6-12 80% or greater of activity at pH optimum;
• (ii) stable pH range stable over a pH range of 6-11 when treated at 40 ° C for 30 minutes;
• (iii) Isoelectric point with an isoelectric point of about 8.9-9.1 and
• (iv) action of a fatty acid a casein-degrading activity which is not inhibited by oleic acid

wherein the amino acid is 90% or more identical to SEQ ID NO: 1 or 2.

In In another aspect of the present invention, one of the above provided gene encoding alkaline protease.

In Yet another aspect of the present invention is a deposited microorganism according to the invention, which will be described hereinafter and that described above produced alkaline protease.

In Yet another aspect of the present invention is a Detergent composition containing the alkaline protease described above.

Short description of drawings

1 shows the effects of pH on the activity of the alkaline protease KP43. 2 shows the effects of pH on the stability of the alkaline protease KP43 (40 ° C, 30 minutes). 3 shows the effects of pH on the stability of alkaline protease KP43 (10 ° C, 24 hours). 4 shows the effects of temperature on the activity of alkaline protease KP43. 5 shows the effects of temperature on the stability of the alkaline protease KP43. 6 shows the effect of an oxidizing agent (50 mM hydrogen peroxide) on the activity of the alkaline protease KP43. 7 shows the N-terminal sequences of protease KP9860 and partially degraded products thereof. 8th shows primer sequences derived from an N-terminal sequence of protease KP9860. 9 shows PCR-amplified fragments of 57 bp and the primer designs.

Best way to execution the invention

The alkaline according to the invention Protease has the above-described physical / chemical Properties (i) to (iv). Of these, feature (iv) is extra important. The alkaline protease has a casein-degrading activity in the presence of 10mM oleic acid, a component of sebum that is as high as in the absence of oleic acid.

The alkaline according to the invention Protease preferably has (v) an estimated molecular weight of about 43,000 as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

• (vi) Wirkungstemperatur und optimale Temperatur wirkend bei einer optimalen Temperatur von 60°C–70°C und ebenfalls wirkend bei einer so niedrigen Temperatur von 20°C oder niedriger;
• (vii) Wirkungen von Metallionen eine Aktivität, die durch Hg²⁺ und Cu²⁺ gehemmt wird und eine Hitzestabilität, welche durch Ca²⁺ verstärkt wird;
• (viii) Wirkungen von Hemmstoffen eine Aktivität, die nicht durch Ethylendiamintetraessigsäure (EDTA) und p-Chloroquecksilberbenzoesäure (PCMB) gehemmt wird und eine Aktivität, die durch Diisopropylfluorphosphat (DFP) und Phenylmethansulfonylfluorid (PMSF) gehemmt wird und
• (ix) Wirkungen von oberflächenaktiven Mitteln eine Aktivität, die nicht durch lineares Natriumalkylbenzolsulfonat, Natriumpolyoxyethylenalkylsulfat, Natriumdodecylsulfat, Natrium-α-Olefinsulfonat oder α-Sulfofettsäureester gehemmt wird.

Particularly preferred is an alkaline protease having, in addition to the properties (i) to (v), properties (vi) to (ix) as described below.

• (vi) action temperature and optimum temperature acting at an optimum temperature of 60 ° C-70 ° C and also acting at such a low temperature of 20 ° C or lower;
• (vii) effects of metal ions, activity inhibited by Hg ²⁺ and Cu ²⁺ and heat stability enhanced by Ca ²⁺ ;
• (viii) effects of inhibitors an activity which is not inhibited by ethylenediaminetetraacetic acid (EDTA) and p-chloro-mercuricbenzoic acid (PCMB) and activity inhibited by diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF) and
• (ix) Effects of surfactants An activity not inhibited by linear sodium alkyl benzene sulfonate, sodium polyoxyethylene alkyl sulfate, sodium dodecyl sulfate, sodium α-olefin sulfonate or α-sulfofatty acid ester.

The alkaline according to the invention Protease preferably has an amino acid sequence shown in Sequence No. 1 or 2 or such a sequence in which one or more amino acids are deleted, substituted or added are. Xaa in sequences Nos. 1 and 2 refers to any one Amino acid. preferred amino acids for Xaa at each position in sequence # 2 are shown in the following table shown:

table

deletions, Substitutions and additions in the alkaline according to the invention Proteases are not particularly limited. Those in sequence no. 1 or 2 shown amino acid sequence However, it is at an altitude of Conserved 90% or more.

Examples of alkaline proteases include alkaline proteases with a Amino acid sequence that in Sequence No. 3, 4 or 5.

(fortgesetzt in Tabelle 1-b)The alkaline protease of the present invention can be produced by culturing the alkaline protease-producing microorganism belonging to the genus Bacillus and collecting the enzyme from the culture broth. Examples of alkaline protease-producing microorganisms according to the present invention include wild strains belonging to the genus Bacillus and a transformant containing a gene encoding a peptide having the above-described amino acid sequence. Examples of the wild strains include KP-43, KP-1790 and KP-9860. Mycological characteristics of these strains are shown below. Table 1-a

(continued in Table 1-b)

Table 1-b

Based on the mycological characteristics described above the three tribes with Reference to the relevant Descriptions in "Bergey's Manual of Systematic Bacteriology "(Williams & Wilkins Co., 1984) and believed to be of the genus Bacillus belong. These tribes however, were new microorganisms in that the characteristics of these species not completely those known species belonging to the genus Bacillus correspond. Therefore were the three tribes at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, 305-0046, JAPAN) as Bacillus sp. KSM-KP43 (FERM BP-6532), Bacillus sp. KSM-KP1790 (FERM BP-6533) and Bacillus sp. KSM-KP9860 (FERM BP-6534) deposited (Date of original Deposit: September 18, 1996).

Around the inventive alkaline Produce protease using the strains described above, become the tribes in a medium containing an assimilable carbon source, a Nitrogen source and essential nutrients, vaccinated and by means of a usual one Method bred.

The Collecting and purifying an alkaline target protease from the thus obtained culture broth can according to conventional Method applicable to collection and cleaning of conventional ones Enzymes, performed become. For example, the cells are centrifuged or Filtration separated from the culture broth and alkaline target protease can by a conventional purification method from the supernatant to be obtained. The enzyme liquid thus obtained can be used as such be further purified and crystallized by a known method become.

alternative can the inventive alkaline Protease can be prepared by the following steps: Obtained a gene encoding the alkaline protease, producing a recombinant vector using the gene, transform a host cell using the recombinant vector, cultivating the obtained transformant and collecting the alkaline target protease from the bred Product.

The gene encoding the alkaline protease of the present invention may be cloned from any of the strains described above. The cloning can be carried out by known methods the. Examples of these methods include (1) the shotgun method comprising preparing a DNA fragment by completely or partially cleaving the chromosomal DNA using an appropriate restriction endonuclease, combining the fragment into a suitable vector, and expressing by introducing into Escherichia coli or Bacillus subtilis and ( 2) a method comprising the synthesis of a suitable primer and the cloning of a target gene by PCR.

Examples the nucleotide sequence of the alkaline protease of the invention in Sequences Nos. 3 to 5. The nucleotide sequence is not restricted to sequence Nos. 3 to 5 and acceptable sequences can a nucleotide sequence encoding the amino acid sequence shown in sequence Nos. 1 or 2, and a nucleotide sequence encoding such an amino acid sequence, in which one or more amino acids are deleted substituted or added are, include. Of these, nucleotide sequences are generated by Sequences Nos. 3 to 5 are shown, or such sequences, in which one or more amino acids are deleted substituted or added are preferred. In these cases occurs deletion, substitution or addition preferably within the Variation of the amino acid sequence described above.

Around a recombinant vector comprising the above-described Gen, which encodes an alkaline protease, can the gene into any vector that is suitable for the gene be expressed in a host of interest. Examples for vectors include pUC18, pBR322 and pUC19 in the event that Escherichia coli serves as a host, and pUB110 in the case of Bacillus subtilis serves as a host.

One Host is produced using the recombinant vector thus obtained with a usual Methods such as the protoplast method or the method of transformed into competent cells. Although no specific restriction for the host applies, microorganisms are preferred. Examples include Gram-positive Bacteria such as microorganisms belonging to the genus Bacillus, Gram-negative Bacteria such as Escherichia coli, yeasts belonging to Saccharomyces and belonging to Aspergillus Mushrooms.

Around the inventive alkaline Protease by breeding To produce the obtained transformant, the breeding, the collecting and the Cleaning in accordance performed with a procedure which is applied in the case where the one described above Wild strain is used.

As As described above, the alkaline protease of the present invention has a excellent resistance to alkaline conditions and possesses excellent protease activity even in the presence of lipids. Therefore, the alkaline protease useful for a Enzyme incorporated in a variety of detergent compositions.

It There is no special restriction in terms of the amount of alkaline protease described above, the is incorporated in a detergent composition, and the amount is preferably 0.1-5000 U based on 1 kg, more preferably 1-500 U of the detergent composition.

Known Detergent ingredients can in the detergent composition of the invention containing the alkaline protease can be incorporated. For example, the Ingredients described in WO94 / 26881 (page 5 top right column, line 14, lower right column, line 29) are applied become.

A surfactants Substance is added to the detergent composition in an amount of 0.5-60% by weight (hereinafter simply referred to as "%"), especially preferably 10-45%, in a powdery detergent composition and in an amount of 20-50% in a liquid Detergent composition incorporated. When a detergent composition of the invention as a bleaching detergent composition or as a detergent composition for one automatic dishwasher serves, becomes a surface-active Substance is typically incorporated in an amount of 1-10%, preferably 1-5%.

One catcher for bivalent Metal ions are incorporated in an amount of 0.01-50%, preferably 5-40%.

One alkaline agent and an inorganic salt are in abundance from 0.01 to 80%, preferably 1-40%, incorporated.

An anti-redeposition agent is incorporated in an amount of 0.001-10%, preferably 1-5% porated.

The Detergent composition may be an enzyme other than the alkaline of the invention Protease included. Examples include cellulase, amylase, protopectinase, Pectinase, lipase, hemicellulase, β-glucosidase, glucose oxidase and cholesterol oxidase. These enzymes are present in an amount of 0.001-5%, preferably 0.1-3% incorporated.

One Bleaching agent such as hydrogen peroxide or percarbonate is preferred in an amount of 1-10% incorporated. If a bleach is incorporated, a Bleach activator can be incorporated in an amount of 0.01-10%.

Examples for fluorescent agents, which are incorporated in the composition include a biphenyl compound like Cinopearl CBS-X and a stilbene compound like a fluorescer of the DM type. The fluorescer is preferably in an amount from 0,001-2% incorporated.

The The detergent composition described above can be used to form a Variety of shapes like liquid, powdery and granular are processed. The detergent composition can be used for laundry, a automatic dishwasher, Drain lines and bits are used and can be used as a bleach become.

Examples

Example 1 (Screening after alkaline protease-producing microorganisms)

A soil sample (1 g) was suspended in physiological saline (10 ml) and thermally treated at 80 ° C for 10 minutes, followed by inoculation into a liquid enrichment medium for protease-producing microorganisms, the medium having the following composition to help with it 20 ° C to breed. After subculture replenishment was repeated approximately three times in the same medium, the cultured product was smeared on a dish to assess protease production and grown at 20 ° C for 5-7 days. The colonies around which a transparent zone was formed by dissociation of the skim milk were selected for the recovery of the protease-producing microorganisms. By the above method, strain Bacillus sp KSM-KP43, strain KSM-KP1790 and strain KSM-KP9860 were obtained as alkaline protease-producing microorganisms. Table 2 Composition of the liquid enrichment medium for the pH11 survey Monopotassium phosphate 0.1% magnesium sulfate 0.02% Yeast extract (Difco) 0.05% Keratin (Tokyo Kasei) 1.0% glucose 0.5% sodium 0.3% Agar plate medium for the screening Nutrient Agar (Difco) 2.3% Skim milk (Difco) 0.3% sodium 1.0%

Example 2

The strain Bacillus sp KSM-KP43 obtained in Example 1 was transformed into a liquid medium comprising polypeptone S (1%), yeast extract (0.05%), potassium phosphate (0.1%), magnesium sulfate (0.02%), glucose ( sterilized separately) (1%) and sodium carbonate (separately sterilized) (0.5%) to be cultured at 30 ° C for 24 hours. The concentration of enzyme in the supernatant fluid was about 1.5 U / L. The supernatant fluid, which was separated from the cells by centrifugation at 4 ° C, was added with stirring powdered ammonium sulfate so that a 90% saturated concentration was reached. The solution was kept stirring at 4 ° C for a whole day and night, and the resulting precipitate was recovered by centrifugation. The resulting precipitate was dissolved in 10 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 5 mM calcium chloride, followed by dialysis through the buffer solution. Subsequently, the dialyzed liquid was applied to a DEAE-Sepharose FF column (product of Pharmacia) equilibrated with 10 mM of a Tris-hydrochloric acid buffer solution (pH 7.5) containing 5 mM calcium chloride to thereby recover the unabsorbed To win the faction. The fractionated liquid was dialyzed through 50 mM HEPES buffer solution (pH 7.5) containing 2 mM calcium chloride and applied to an SP-Sepharose FF column equilibrated with the same buffer solution to thereby collect an active fraction , which eluted slightly after the unabsorbed fraction. While the active fraction having a recovery rate of 15% was used as a sample, SDS-polyacrylamide electrophoresis was performed, and as a result, a single band was obtained for the corresponding enzyme.

Example 3

The obtained strains Bacillus sp KSM-KP1790 and KSM-KP9860 were in the same medium as grown in Example 2 and the alkaline protease was in the same way as in Example 2 cleaned.

Example 4

The enzymatic properties of those obtained in Examples 2 and 3 alkaline proteases were examined. The procedures and results The experiments are described below.

I. Materials and Methods for the experiments

(1) Method of measuring activity

(a) Method in which Casein is used as a substrate

After this 1 ml of 50 mmol / l of different buffer solutions containing 1% (w / v) Casein (Hammerstein: product of Merck Inc.), at 40 ° C for 5 minutes 0.1 ml of an enzyme solution was added to the solution, followed by incubation at 40 ° C for 10 Minutes. 2 ml of a TCA solution (0.11 mol / l trichloroacetic acid: 0.22 mol / l sodium acetate: 0.33 mol / l acetic acid) were added to give the Stop reaction, and the mixture was left at room temperature for 10 minutes ditched. Subsequently it was acid denatured protein filtered (# 2 filter paper: product of Whatman). To 0.5 ml of the filtrate was added 2.5 ml of an alkaline Copper reagent (1% (w / v) sodium potassium tartrate: 1% (w / v) copper sulfate: 2% (w / v sodium carbonate, 0.1 mol / l sodium hydroxide = 1: 1: 100 (V / v) and, after keeping the solution at 30 ° C for 10 minutes 0.25 ml was diluted Phenol reagent (phenol reagent (product of Kanto Chemical) in duplicate dilute with deionized water) and after leaving for 30 minutes at 30 ° C was held, the solution became subjected to absorption measurement at 660 nm. The following solution became used as a blank: to the system described above Enzyme reaction, a reaction stop solution was mixed and then became the enzyme solution added.

A Unit (P.U.) of enzymatic activity was considered the amount of enzyme defines which acid-soluble protein degradation products, corresponding to 1 mmol of tyrosine per minute under the above reaction conditions unleashed.

(b) Method in which a synthetic oligopeptide is used as the substrate

0.05 ml of a 50 mmol / l synthetic oligopeptide solution (succinyl-alanyl-alanyl-prolyl-leucine-paranitroanilide, solved in dimethyl sulfoxide) were dissolved in 0.9 ml of a 100 mmol / L boric acid buffer solution (pH 10.0, containing 2 mmol / l calcium chloride) and, after the solution for 5 minutes at 30 ° C 0.05 ml of an enzyme solution was added followed by Incubation at 30 ° C for 10 Minutes. 2 ml of a 5% (w / v) citric acid was added, to stop the reaction and the absorbance became 420 nm measured.

One unit (U) of enzymatic activity was defined as the amount of an enzyme which was acid Released protein degradation products, corresponding to 1 mmol of tyrosine, per minute under the above reaction conditions released.

(c) Method in which hemoglobin is used as a substrate

According to the method of Anson (ML Anson, J. Gen. Physiol., 22, 79 (1983)), hemoglobin from bovine blood serum was denatured using urea and adjusted to pH 10.5 with sodium hydroxide. 0.1 ml of an enzyme solution (1.0 × 10 ^-5 -1.0 × 10 ^-3 AU) was added to 0.5 ml of the substrate solution (2.2% based on hemoglobin), and the resulting solution was allowed to stand for 10 minutes incubated at 25 ° C. To the resulting solution was added 1.0 ml of 4.9% trichloroacetic acid to stop the reaction. After completion of the reaction, centrifugation (3,000 rpm, 10 minutes) was performed, and the protein degradation products in the supernatant liquid were quantitatively determined according to the Folin-Lowry method (OH Lowry et al., J. Biol. Chem., 193, 265 (1951 )).

A Unit (A U) enzymatic activity was considered the amount of enzyme defines which acid-soluble protein degradation products, corresponding to 1 mmol of tyrosine per minute under the above reaction conditions unleashed.

(2) Optimal pH

0.1 ml of an enzyme solution (3.0 x 10 ^-5 mP.U.) was added to 1 ml of a 50 mmol / l Britton-Robinson buffer solution containing 1% (w / v) casein and the activity was measured according to the casein method.

(3) pH stability

An enzyme solution (8.0 × 10 ^-4 mP.U.) was mixed in a Britton-Robinson buffer solution (20 mmol / L containing 2 mmol / L calcium chloride), followed by treatment at 40 ° C for 30 minutes or at 10 ° C for 24 hours. After cooling on ice, the treated solution was diluted 40-fold with 50 mmol / L boric acid buffer solution, followed by measurement of residual activity according to the method in which casein is used as a substrate.

(4) Optimal temperature

0.1 ml of the enzyme solution (2.0 × 10 ^-5 mP.U.) was added to 1 ml of a 50 mmol / l boric acid buffer solution (pH 10.0) containing 1% (w / v) casein, and the activity of the enzyme was measured at temperatures between 10 and 80 ° C according to the casein method.

The activity measurements were performed in both systems, i. H. in the presence and in the absence of 5 mmol / L calcium chloride.

(5) heat stability

An enzyme solution (2.5 × 10 ^-4 mP.U.) was added to 20 mmol / L boric acid buffer solution (pH 10.0) in both systems, ie in the presence and absence of 5 mmol / L calcium chloride, and thermally treated at the appropriate temperature for 10 minutes. After being cooled with ice, the treated solution was diluted 5 times with 50 mmol / L boric acid buffer solution (pH 10.0) and the residual activity was measured using casein as a substrate.

(6) Effects of metal ions

An enzyme solution (4.0 × 10 ^-4 mP.U.) was added to 20 mmol / L boric acid buffer solution (pH 10.0) containing 1 mmol / L various metal salts, and the resulting solution was allowed to stand for 20 minutes Incubated at 30 ° C. The solution was diluted 5 times with 50 mmol / L boric acid buffer solution (pH 10.0), followed by measurement of the activity using casein as a substrate.

(7) Effects of inhibitors

The enzyme solution (1.0 × 10 ^-3 mP.U.) was added to 10 mmol / L of phosphoric acid buffer solution (pH 7.0) containing various inhibitors so as to obtain a predetermined concentration, and the Solution was incubated for 20 minutes at 30 ° C. Then, the solution was diluted 20-fold with deionized water, and the residual activity was measured using casein as a substrate.

(8) Effects of surfactant substances

An enzyme solution (7.0 × 10 ^-4 mP.U.) was added to 100 mmol / L boric acid buffer solution containing dissolved surfactants in an amount of 1%, and the resultant solution was incubated at 40 ° C for 4 hours , The solution was diluted 20-fold with 50 mmol / L boric acid buffer solution (pH 10.0), and the residual activity was measured using casein as a substrate.

(9) Effects of oxidizing Means (hydrogen peroxide)

2.7 ml of a Britton-Robinson buffer solution containing hydrogen peroxide and calcium chloride (final concentration: 50 mmol / l hydrogen peroxide, 2 mmol / L calcium chloride, 2 mmol / L Britton-Robinson) (pH 8.0) for 15 Minutes at 30 ° C and then 0.3 ml of an enzyme solution was added. Over time a sample of 0.8 ml of the resulting solution in a previously prepared Test tubes, containing 5 μl Catalase (Boehringer Mannheim Co .: 20 mg / L) to thereby to stop the oxidation reaction. Each sample was suitable Were diluted with 2 mmol / l calcium chloride and the residual activity was according to the method, in which synthetic oligopeptide is used as a substrate, measured.

(10) Effects of fatty acids

Under Use of 50 mM phosphoric acid buffer solution (pH 7) containing 1% (w / v) casein as substrate solution a reaction in the presence of 0-10 mM sodium oleate for 15 minutes executed at 20 ° C and the activity was measured using casein as a substrate.

II. Results

(1) Optimal pH

The effects of pH on three types of proteases (KP43, KP1790 and KP9860) were investigated. 1 Figure 2 shows activity (100%) of KP43 normalized in activity at optimum pH at each pH, indicating that the optimal pH range of proteases of the invention is 6-12. Therefore, these enzymes show a high protein degrading activity in the very broad pH range.

(2) pH stability

After allowing it to stand at 40 ° C for 30 minutes or at 10 ° C for 24 hours, the residual activity of KP43 was measured over a range of pH's. The 2 and 3 show the residual activities that were normalized with respect to enzyme activity before treatment (100%). The results show that the enzymes according to the invention are stable over a pH range of 6-12 after treatment for 30 minutes at 40 ° C. and that the addition of calcium ions improves the enzyme stability at pH 5. Similarly, the results indicate that the enzymes of the present invention are stable over a broad pH range of 5-12 after treatment for 24 hours at 10 ° C.

(3) Optimal temperature

Using casein as a substrate, the effects of temperature on the proteases were examined. 4 shows the activities of KP43 over a temperature range normalized with respect to the highest activity in the absence of calcium ions (100%). The results show that for all three types of proteases in the absence of calcium ions the optimum temperature is 60 ° C and in the presence of calcium ions 70 ° C. Therefore, the results show that the optimum temperature is shifted up by the addition of calcium ions, as is the case with conventional proteases for a detergent.

(4) heat stability

The heat treatment was carried out for 10 minutes at temperatures in the range of 30-80 ° C (pH 10.0 in the presence and in the absence of 5 mmol / l calcium chloride) and the residual activity was measured. 5 shows the residual activity of KP43 at each treatment temperature, normalized in terms of activity before treatment (100%). The results show that the proteases are stable in the absence of calcium chloride at temperatures of up to 60 ° C and that the addition of calcium chloride (5 mmol / L) has the effect of shifting the temperature stability upwards by about 10 ° C. In comparison with commercially available detergent enzymes, these enzymes have a high temperature stability, namely a stability comparable to that of esperase, which has the most excellent temperature stability among the commercially available enzymes.

(5) Effects of metal ions

Behandlungsbedingungen: 1 mM Metallsalz, 20 mM Boratpuffer (pH 10,0), 30°C, 20 MinutenIn a 20 mmol / L boric acid buffer solution (pH 10), 3 kinds of proteases were treated with various metal salts (1 mmol / L) for 20 minutes at 30 ° C, and the residual activity was measured. The residual activity was normalized (100%) with respect to the enzyme activity obtained for the protease treated in the same manner except for the addition of metal salts (see Table 3). The results show that the activity is inhibited by mercuric chloride and silver nitrate, but that the activity is extremely stable to other metal salts. Table 3

Treatment conditions: 1 mM metal salt, 20 mM borate buffer (pH 10.0), 30 ° C, 20 minutes

(6) Effects of metal ions

EDTA:

Ethylendiamintetraessigsäure (Sigma)

EGTA:

Ethylenglycoltetraessigsäure (Sigma)

DTT:

Dithiothreit (Sigma)

PCMB:

p-Chloroquecksilberbenzoat (Sigma)

NEM:

N-Ethylmaleimid (Sigma)

DFP:

Diisopropylfluorphosphorsäure (Sigma)

PMSF:

Phenylmethansulfonylfluorid (Sigma)

The effects of general enzyme inhibitors on the alkaline proteases of the invention were investigated. A variety of inhibitors were added to 10 mmol / L phosphoric acid buffer solution (pH 7.0) to reach the predetermined concentration, and the resulting solution was incubated at 30 ° C for 20 minutes, after which the residual activity was measured. The residual activity was normalized with respect to the enzyme activity obtained for the protease treated in the same manner as described above in the absence of inhibitors (100%) (see Table 4). The results show that activity was inhibited for all three types of proteases by diisopropylfluorophosphoric acid (DFP), phenylmethanesulfonyl fluoride (PMSF) and chymostatin, which are known serine protease inhibitors. Therefore, the proteases of the present invention are believed to have a serine residue in their active site. In contrast, effects of antipin and leupeptin from actinomycetes reported to inhibit serine proteases have not been found. Table 4

EDTA:

Ethylenediaminetetraacetic acid (Sigma)

EGTA:

Ethylene glycol tetraacetic acid (Sigma)

DTT:

Dithiothreitol (Sigma)

PCMB:

p-Chloro mercuric benzoate (Sigma)

NEM:

N-ethylmaleimide (Sigma)

DFP:

Diisopropylfluorophosphoric acid (Sigma)

PMSF:

Phenylmethanesulfonyl fluoride (Sigma)

(7) Effects of surfactant substances

Behandlungsbedingungen: 1% grenzflächenaktive Substanz, 100 mM Boratpuffer (pH 10,0), 40°C, 4 StundenEach protease was treated with a variety of 1% surfactants for 4 hours at 40 ° C in a 0.1 mol / l tris-hydrochloride buffer solution (pH 9.0) and the residual activity was measured. The residual activity is normalized in terms of enzyme activity in the case of no treatment (100%) (see Table 5), indicating that the three kinds of surface-active-enzyme-inhibiting agents characterized by linear alkylbenzenesulfonic acids (LAS) are extremely stable , Accordingly, the enzymes were considered to be a useful ingredient of surfactant-containing detergents. Table 5

Treatment conditions: 1% surfactant, 100 mM borate buffer (pH 10.0), 40 ° C, 4 hours

(8) Effects of oxidizing substances

Each protease was treated at 30 ° C in 50 mmol / L Britton-Robinson buffer solution containing hydrogen peroxide (pH 8.0), and the residual activity was measured as a function of time. As in 6 KP43 showed a much higher stability than that of commercially available Savinase or KAP and showed a stability as high as that of Durazym (Novo Nordisk), which was achieved by conferring resistance to Savinase oxidizing agents using protein manipulation techniques was developed.

(9) Effects of fatty acids

As shown in Table 6, the activity of the alkaline Proteases not by oleic acid, one of the components of sebum, inhibited.

Table 6 Relative Activity (%) in the Presence of Fatty Acid

Example 5 (Cloning a gene encoding the KP9860 protease)

(1) Preparation of a genomic DNA of KSM-KP9860

Strain KSM-KP9860 was grown at 30 ° C for two days in a liquid medium (0.5% glucose, 0.2% polypeptone-S, 0.05% yeast extract, 0.1% KH ₂ PO ₄ .7H ₂ O, 0.26% NaCO ₃ : pH 9.0) (500 ml) and the cells were collected by centrifugation. Genomic DNA was prepared from the obtained cells by the method of Saito and Miura (Biochim Biophys Act 72, 619 (1963)). (2) Limited proteolysis of KP9860 protease 1) Denaturation of KP9860 protease KP9860 protease (5 mg / ml) 45 μl PMSF (100mM) 20 μl EDTA (200mM) 10 μl SDS (0.08 mg / ml) 25 .mu.l

A protease solution having the above composition was heated in boiling water for 10 minutes. The protease solution was dialyzed against ammonium acetate (2 mM) to thereby remove SDS, EDTA and PMSF and was then lyophilized. Subsequently, the lyophilized protease was dissolved in distilled water (100 μl) to thereby serve as a sample of denatured protein. 2) Limited proteolysis by trypsin Denatured protein sample 100 μl Trypsin (1 μg / ml, Sigma) 100 μl 1 M Tris-HCl (pH 7.5) 50 μl Distilled water 750 μl

trypsin with the denatured protein prepared in 1) in a Ice bath for 3 hours in the solution react with the above composition. After adding 300 μl SDS (0.08 mg / ml), 100 μl EDTA (200mM) and 200μl PMSF (100 mM) was limited in proteolysis by heating for 3 minutes in finished boiling water.

SDS, EDTA and PMSF were removed by dialysis against ammonium acetate (2 mM) and the solution became lyophilized. Subsequently The lyophilizate was dissolved in distilled water (100 μl) to thereby as a sample for SDS-PAGE to serve.

3) Extraction of the partial degraded product

The in 2) was subjected to SDS-PAGE with 12% Ready-Gel-J (product from Bio-Rad). The protein bands were stained with a Quick-CBB staining solution (product from Bio-Rad). The gel containing the protein band was cut with a razor blade and the gel piece became in a 1.5 ml tube in pieces pound. The buffer for SDS-PAGE (composition: glycine 14.4% (w / v), Tris 3.03%, SDS (Bio-Rad product) 10%) was dissolved in 5 volumes of crushed gel and the mixture was stirred at room temperature to thereby eluting the protein band. The eluate was against ammonium acetate (2 mM) and then lyophilized. The lyophilized sample served for the protein sequencer Type 476A (product of Applied Biosystem), to determine the N-terminal sequence.

The obtained N-terminal sequences are described in 7 shown.

(3) PCR

20-30 nucleotides primers for the 5 'end of the + chain and that of the - chain, corresponding to the N-terminal sequences obtained, were synthesized. The PCR reaction was carried out in a 100 μl reaction system using a template DNA (100 ng), a primer (20 pmol) and PwoDNA polymerase (product of Boehringer Mannheim). When reverse PCR was performed, the Expand ^™ longtemplate PCR system (product of Boehringer Mannheim) was used in a 50 μl reaction system. A PCR performed using these primers 9860-N2 and 9860-25k RV yielded a fragment of 527 bp.

(4) Subcloning of the PCR product

The PCR product was purified with a High Pure PCR Product Purification Kit (product of Boehringer Mannheim) and into the Sma I site of pUC18 by reaction overnight at 16 ° C with the ligation kit Ver. 2 (product of Takara) inserted. The resulting recombinant plasmid and the competent cells of strain E. coli JM109 (product of Takara) were mixed and the mixture became a hit zeschock (42 ° C, 45 seconds) to thereby transform the E. coli JM109 cells. LB was added to the cells. After being kept at 37 ° C for one hour, the mixture was applied to an LB plate containing IPTG (0.1 mM, Sigma), X-GaI [0.004% (w / v), Sigma] and ampicillin (50 μg / ml, Sigma). The cultivation was carried out overnight at 37 ° C, and grown white colonies were selected as those transformants carrying the recombinant plasmid.

(5) Determination of nucleotide sequence

The Transformant was over Night at 37 ° C in LB containing ampicillin (50 μg / ml), cultured and the cells were collected by centrifugation. The recombinant Plasmid was prepared using the High Pure Plasmid Isolation Kit (Product of Boehringer Mannheim). The PCR for sequencing was in a 20 μl reaction system using a primer and a DNA sequencing kit (product of PERKIN ELMER); the resulting recombinant plasmid (1 μg) served as template DNA. The reaction product was purified using a Quick Spin Column (Product of Boehringer Mannheim) and using dried an evaporation centrifuge. The sample thus treated was an analysis using a DNA Sequencer Type 377 (product from Applied Biosystem).

The DNA fragment obtained by PCR had the amino acid sequence, which matches the N-terminal sequence of the KP-9860 protease, and there were observed sequences, with general sequences close to Asp and His for three amino acids (Asp, His, Ser), which form an active center of alkaline proteases such as subtilisin. Therefore, from the DNA fragment assumed that it is part of the KP-9860 protease gene.

(6) Southern hybridization

The KP9860 chromosome was digested with EcoRI, SacI, KpnI, HindIII, BamHI, XhoI, Treated PstI and BglII. Southern hybridization was carried out Using the obtained 527 bp DNA as a probe to thereby a complementary one Prove the region.

When Hybridization bands were found in tracks other than the result belonging to KpnI Track observed.

(7) reverse PCR

Inverse PCR was performed using primers (1 ~ 4 ( 9 ) synthesized from the obtained 527 bp sequence. The KP-9860 chromosome was completely cleaved using restriction enzymes, ie EcoRI, HindIII, PstI and BglII and each sample was probed using the Ligation Kit Ver. 2 (product of Takara) for ring closure. Each of the resulting reaction mixtures served as a template DNA for the reverse PCR. The PCR reaction (conditions: (94 ° C-10 seconds, 60 ° C-30 seconds, 68 ° C-4 minutes) × 10 cycles (94 ° C-10 seconds, 60 ° C-30 seconds, 68 ° C-4 minutes + 20x the number of cycles) × 20 cycles; 68 ° C-7 minutes and 4 ° C-1 minute) was performed using the above-described template DNA (0.1 μg), primers 1 and 4 (10 pmol each) and the Expand Long Plate PCR System. In addition, PCR (conditions as described above) was performed using the template DNA from the Eco RI digested chromosome (0.1 μg), primers 2 and 3 (10 μM each), and the Expand Long Plate PCR System. The resulting amplified DNA fragments were purified using the High Pure PCR Product Purification Kit and the ends were blunt-ended using the DNA Blunting Kit (product of Takara). Each of the resulting DNA fragments and the SmaI digested pUC18 were mixed and the mixture was mixed with the Ligation Kit Ver. 2 treated. As described above, E. coli strain JM109 was transformed by the recombinant plasmid, and the resulting recombinant plasmid served as a template DNA for sequencing. Thus, the nucleotide sequence of the amplified DNA fragments was determined.

(8) analysis of the whole Nucleotide sequence of the KP-9860 protease gene

Sequencing revealed that the KP-9860 protease gene contains an open reading frame (ORF) encoding the 1917 bp, 639 amino acid residues and that the ORF contains a region (NDVARHIVKADVAQSSYGLY) that matches the N-terminal sequence of the purified KP9860 protease , Starting from the N-terminal sequence, it was deduced for the mature region of the KP9860 protease gene consisting of 1302 bp encoding 434 amino acids (Sequence No. 3, molecular weight 45310 Da). Sequences upstream of the ORF were observed to be a promoter region (-35 region: ttgtgt, -10 region: tacgat) and a ribosome binding site (aggagt). Downstream of the stop codon (taa), there was a reverse repeating sequence with a free energy of -26.2 kcal / mole, which is believed to be a terminator.

The The procedure of Example 5 was repeated to thereby evaluate the entire Nucleotide sequence and the amino acid sequence to analyze each of the genes of KP-43 protease and KP-1790 protease. The results are shown in Sequences Nos. 4 and 5.

Example 6

Washing test:

A washing test was performed according to JIS K 3371. Detergents, the compositions of which are shown in Table 7, are dissolved in water containing 71.2 mg CaCO ₃ / l (4 ° DH) to adjust the concentration and each protease added to the detergent solution to determine the concentration of alkaline protease to 40 mAPU / l according to the Anson hemoglobin method (see Table 8).

• *) G steht für granulär.
• **) L steht für flüssig.

LAS:

Lineares Natriumalkyl(C12-C14)benzolsulfonat (freie Säure in ein flüssiges Detergens inkorporiert)

AS:

Alkylsulfat

AE:

Polyoxyethylenlaurylether (durchschnittliche EO-Zugabe von 4 Mol)

AEP:

Polyoxyethylen-Polyoxypropylenlaurylether (durchschnittliche EO-Zugabe von 8 Mol, durchschnittliche PO-Zugabe von 3 Mol))

AES:

Alkylethersulfat (durchschnittliche EO-Zugabe von 2,5 Mol)

Fettsäure:

Natriumsalz der Fettsäure aus Palmöl

Zeolit:

Zeolit 4A, durchschnittliche Partikelgröße von 3 μm

Natriumcarbonat:

dichte Asche

Amorphes

Silikat: JIS Nr. 2-Natriumsilikat

Kristallines

Silikat: pulverisiertes SKS-6 (Produkt von Hoechst Tokuyama), durchschnittliche Partikelgröße von 15 μm

AA-MA:

Sokalan CP5, Acrylsäure-Maleinsäure-Kopolymer (Produkt von BASF)

PEG:

Polyethylenglycol, durchschnittliches Molekulargewicht von 8.000

Collar shirts (worn for 3 days) were used as samples. For comparison, after the cloth of a collar was cut into the size of 8 × 8 cm, the cloth was set at 15 ° C and 100 rpm for 10 minutes using a Terg-O-Tometer (Ueshima Seisakusyo) with addition of the enzyme or without Addition of the enzyme washed. After being rinsed and dried, pairs of neckties (15 pairs) were compared and rated by visual assessment. When the soil was almost completely cleaned, a rating of 5 was assigned and if the soil was barely cleaned, a rating of 1 was assigned and the total scores of 15 samples were calculated. The detergency index was expressed as the score of each composition, assuming the detergency of a detergent composition without addition of enzyme as 100%. The results are shown in Table 8. Table 7

• *) G stands for granular.
• **) L stands for liquid.

READ:

Linear sodium alkyl (C12-C14) benzenesulfonate (free acid incorporated in a liquid detergent)

AS:

alkyl sulfate

AE:

Polyoxyethylene lauryl ether (average EO addition of 4 moles)

AEP:

Polyoxyethylene-polyoxypropylene lauryl ether (average EO addition of 8 mols, average PO addition of 3 mols))

AES:

Alkyl ether sulfate (average EO addition of 2.5 moles)

Fatty acid:

Sodium salt of fatty acid from palm oil

zeolite:

Zeolite 4A, average particle size of 3 μm

Sodium:

dense ash

amorphous

Silicate: JIS No. 2 sodium silicate

crystalline

Silicate: powdered SKS-6 (product of Hoechst Tokuyama), average particle size of 15 μm

AA-MA:

Sokalan CP5, acrylic acid-maleic acid copolymer (product of BASF)

PEG:

Polyethylene glycol, average molecular weight of 8,000

Table 8

table Figure 8 shows that even under the same activity conditions, an enzyme of the invention containing detergent composition (Detergent A) a higher detergency compared to detergents containing conventional proteases, having. Detergents B and C are also excellent washing power according to the invention.

Example 7

• *) G steht für granulär.
• **) L steht für flüssig.

LSA-2:

Alkylbenzolsulfonsäure (C10-C14-Alkylkette), welche mit 48-%iger NaOH neutralisiert wurde

LSA-3:

Alkylbenzolsulfonsäure (C10-C14-Alkylkette), welche mit 50-%iger NaOH neutralisiert wurde

AS-2:

Natriumsalz von Dovanol 25-Sulfat (C12-C15-Sulfat)

SAS:

Natrium C13-C18 Alkansulfonat

AOS:

Natrium-α-Olefinsulfonat

SFE:

Natriumsalz eines Palmöl-α-Sulfofettsäuremethylesthers

Fettsäuresalz:

Natriumpalmitat

AES-2:

Natriumpolyoxyethylenalkyl(C12-C15)ethersulfat (durchschnittliche EO-Zugabe von 2 Mol)

AE-3:

EO-Addukt (durchschnittlich 3 Mol) von C12-C13-Alkohol

AE-4:

EO-Addukt (durchschnittlich 7,2 Mol) von C12-C15-Alkohol

AE-5:

EO-Addukt (durchschnittlich 7 Mol) von sekundärem C12-C15 Alkohol

AG:

Alkyl (aus Palmöl)-Glucosid (durchschnittlicher Polymerisationsgrad von 1,5) öl-absorbierender Träger: Amorphes Natriumaluminosilikat, Ölabsorption von 235 ml/100 g

Kristallines Silikat:

SKS-6 (δ-Na₂Si₂O₅, kristallin-geschichtetes Silikat, durchschnittliche Partikelgröße von 20 μm)

Amorphes Silikat:

JIS Nr. 1-Natriumsilikat

STPP:

Natriumtripolyphosphat

NTA:

Natriumnitrilotriacetat

PAA:

Natriumsalz der Poly(acrylsäure), durchschnittliches Molekulargewicht von 12.000

AA-MA:

Acrylsäure/Maleinsäure-Kopolymer

CMC:

Natrium-Carboxymethylcellulose

PEG:

Polyethylenglycol, durchschnittliches Molekulargewicht von 6.000

PVA:

Polyvinylpyrrolidon, durchschnittliches Molekulargewicht von 40.000, K-Wert von 26–35

Fluoreszenzfarbstoff:

Tinopal CBS und Whitex SA (1:1 (Gew.)), nur Cinopearl in ein flüssiges Detergens inkorporiert

Parfüm:

Eine Parfümzusammensetzung, offenbart in der Offengelegten Japanischen Patentanmeldung (kokai) Nr. 8-239700

Enzym:

Lipolase 100T, Termamyl 60T und KAC 500^® (Produkt der Kao Corporation) 1 : 1 : 1 (Gew.)

PC:

Natriumpercarbonat, durchschnittliche Partikelgröße von 400 μm, beschichtet mit Natriummetaborat

AC-1:

Tetraacetylethylendiamin

AC-2:

Natriumlauroyloxybenzolsulfonat

A granular product was prepared by a method disclosed in Japanese Patent Application Laid-open (kokai) No. 62-257990, using a purified sample of the protease of the present invention derived from Bacillus sp. KSM-KP43, KSM-KP1790 or KSM-KP9860 and was prepared as in Example 2 or 3. The granular product (6 APU / g) (1 part by weight) was incorporated into each of the detergents (100 parts by weight) having the compositions shown in Table 9 to thereby obtain the compositions of the present invention. When the detergent was of the granular type, such a detergent was prepared by mixing a granular detergent base devoid of ingredients, ie, an enzyme, PC, AC-1 and AC-2, with a granulated enzyme, granulated PC, granulated AC-1 and granulated AC-2. Each detergent was dissolved in water containing 71.2 mg CaCO ₃ / l (4 ° DH) in a use concentration and a collar was washed in a manner as described in Example 6. The detergents produced herein have excellent detergency and are useful as detergents. Table 9

• *) G stands for granular.
• **) L stands for liquid.

LSA-2:

Alkylbenzenesulfonic acid (C10-C14 alkyl chain), which was neutralized with 48% NaOH

LSA-3:

Alkylbenzenesulfonic acid (C10-C14 alkyl chain), which was neutralized with 50% NaOH

AS-2:

Sodium salt of Dovanol 25 sulfate (C12-C15 sulfate)

SAS:

Sodium C13-C18 alkanesulfonate

AOS:

Sodium α-olefin sulfonate

SFE:

Sodium salt of a palm oil α-sulfofatty acid methyl ester

Fatty acid salt:

sodium palmitate

AES-2:

Sodium polyoxyethylene alkyl (C12-C15) ether sulfate (average EO addition of 2 moles)

AE-3:

EO adduct (average 3 moles) of C12-C13 alcohol

AE-4:

EO adduct (average 7.2 moles) of C12-C15 alcohol

AE-5:

EO adduct (average 7 moles) of secondary C12-C15 alcohol

AG:

Alkyl (from palm oil) glucoside (average degree of polymerization of 1.5) oil-absorbing carrier: amorphous sodium aluminosilicate, oil absorption of 235 ml / 100 g

Crystalline silicate:

SKS-6 (δ-Na ₂ Si ₂ O ₅ , crystalline-layered silicate, average particle size of 20 μm)

Amorphous silicate:

JIS No. 1 sodium silicate

STPP:

sodium tripolyphosphate

NTA:

sodium nitrilotriacetate

PAA:

Sodium salt of poly (acrylic acid), average molecular weight of 12,000

AA-MA:

Acrylic acid / maleic acid copolymer

CMC:

Sodium carboxymethylcellulose

PEG:

Polyethylene glycol, average molecular weight of 6,000

PVA:

Polyvinylpyrrolidone, average molecular weight of 40,000, K value of 26-35

Fluorescent dye:

Tinopal CBS and Whitex SA (1: 1 (wt)), only Cinopearl incorporated in a liquid detergent

Perfume:

A perfume composition disclosed in Japanese Patent Application Laid-Open (kokai) No. 8-239700

Enzyme:

Lipolase 100T, Termamyl 60T, and KAC 500 ^® (product of Kao Corporation) 1: 1: 1 (wt.)

PC:

Sodium percarbonate, average particle size of 400 μm, coated with sodium metaborate

AC-1:

tetraacetylethylenediamine

AC-2:

Natriumlauroyloxybenzolsulfonat

Example 8

• ¹⁾ Partikelgröße: 500–700 μm
• ²⁾ Lineares Natriumalkylbenzolsulfonat (C12-C14)
• ³⁾ Polyoxyethylenalkylether (C12-C14-Alkyl, durchschnittliche EO Zugabe von 12 Mol)
• ⁴⁾ Durchschnittliches Molekulargewicht von 8.000

Among the ingredients shown in Table 10, sodium percarbonate and sodium carbonate (dense ash) were mixed with stirring. To the mixture was added a 40% aqueous solution of sodium polyacrylate and linear sodium alkyl benzene sulfonate (or nonionic surfactant or Sodium lauroylbenzenesulfonate). Subsequently, a granulation product of the alkaline protease, which was prepared from Bacillus sp. KSM-KP43, prepared as in Example 7, was added to the mixture. The resulting mixture was stirred homogeneously to thereby prepare a bleaching agent. A collar was immersed for 30 minutes at 20 ° C in a 0.5% aqueous solution of each bleach and then with detergent A (Example 6) in a Terg-O-Tometer at 100 rpm for 10 minutes at 20 ° C washed. The resulting bleaching agents have excellent bleaching ability and are useful as bleaching agents for laundry. Table 10

• ¹⁾ Particle size: 500-700 μm
• ²⁾ Linear Sodium Alkylbenzenesulfonate (C12-C14)
• ³⁾ polyoxyethylene alkyl ether (C12-C14-alkyl, average EO addition of 12 moles)
• ⁴⁾ Average molecular weight of 8,000

Example 9

• ¹⁾ Polyoxyethylen-Polyoxypropylen-Kopolymer (durchschnittliches Molekulargewicht von 2.000)
• ²⁾ Ethylenoxid (7 Mol) und Propylenoxid (8,5 Mol)-Addukt von sekundärem C12-C14-Alkohol
• ³⁾ JIS Nr. 2-Natriumsilikat
• ⁴⁾ Acrylsäure-Maleinsäure-Kopolymer

The procedure of Example 8 was repeated to thereby prepare the detergent compositions for an automatic dishwasher having a composition shown in Table 11. The detergency of the obtained compositions was tested under the following conditions. The resulting detergents have excellent detergency and are useful as a detergent for an automatic dishwasher. Table 11

• ¹⁾ polyoxyethylene-polyoxypropylene copolymer (average molecular weight of 2,000)
• ²⁾ Ethylene oxide (7 moles) and propylene oxide (8.5 moles) adduct of C12-C14 secondary alcohol
• ³⁾ JIS No. 2 sodium silicate
• ⁴⁾ acrylic acid-maleic acid copolymer

(1) Preparation of a soiled dishes

egg yolk (2.5 g) was uniform a ceramic plate with a diameter of 25 cm brushed. Of the Plate was in a dryer for 60 minutes at 115 ° C dried.

(2) washing conditions

Dishwasher Used: A Fully Automatic Dishwasher (NP-810, Product of Matsushita Electric Industry Co., Ltd.)
Type of washing: standard gear
Washing water: hardness of 62.3 mg CaCO ₃ / l (3.5 ° DH)
Concentration of the detergent: 0.2% by weight

(3) Evaluation procedure

Five polluted Plates were in a dishwasher under the above conditions using detergent compositions washed from Example 9. The washed dishes were with a 1% erythrosine solution colored, to stain the remaining protein. The degree of protein contamination was assessed visually.

Example 10

• ¹⁾ Acrylsäure/Maleinsäure-Kopolymer (Produkt von BASF)
• ²⁾ Ethylenoxid (7 Mol)- und Propylenoxid (4,5 Mol)-Addukt von sekundärem C12-C14-Alkohol
• ³⁾, ⁴⁾ Synthetisches Beispiel, offenbart in der Offengelegten Japanischen Patentanmeldung (kokai) Nr. 6-179899

Detergent compositions for an automatic dishwasher were obtained from the components shown in Table 12. The detergency of these compositions was evaluated by a test similar to that of Example 9. The compositions provided excellent detergency. Table 12

• ¹⁾ acrylic acid / maleic acid copolymer (product of BASF)
• ²⁾ Ethylene oxide (7 moles) and propylene oxide (4.5 moles) adduct of secondary C 12 -C 14 alcohol
• ³⁾ , ⁴⁾ Synthetic Example disclosed in Japanese Patent Application Laid-Open (kokai) No. 6-179899

Example 11

• ¹⁾ Ein granuläres Produkt, hergestellt durch ein Verfahren, offenbart in der Offengelegten Japanischen Patentanmeldung (kokai) Nr. 62-257990, unter Verwendung einer gereinigten Probe der erfindungsgemäßen Protease, welche aus dem Stamm Bacillus sp. KSM-KP 43 stammte und wie in Beispiel 2 (6 APU/g) hergestellt wurde.
• ²⁾ Die Protease K-16 offenbart in der Offengelegten Japanischen Patentanmeldung (kokai) Nr. 5-25492, welche durch ein in der Offengelegten Japanischen Patentanmeldung (kokai) Nr. 62-257990 offenbartes Verfahren so modifiziert war, dass sie 5 APU/g hatte.
• ³⁾ KAC-500^® (Cellulase, 500 U/g, Produkt der Kao Corporation)
• ⁴⁾ Lipolase 100T^® (Produkt von Novo Nordisk)

Enzymes were added to the above-described Detergent A (Example 6) in amounts shown in Table 13 below. A collar portion of a white shirt was washed in a manner similar to that of Example 6. Table 13

• ¹⁾ A granular product prepared by a method disclosed in Japanese Patent Application Laid-open (kokai) No. 62-257990, using a purified sample of the protease of the present invention derived from the strain Bacillus sp. KSM-KP 43 was prepared as in Example 2 (6 APU / g) de.
• ²⁾ Protease K-16 disclosed in Japanese Laid-Open Patent Application (kokai) No. 5-25492 which was modified by a method disclosed in Japanese Patent Application Laid-Open (kokai) No. 62-257990 to be 5 APU / g would have.
• ³⁾ KAC- ^500® (Cellulase, 500 U / g, product of Kao Corporation)
• ⁴⁾ Lipolase 100T ^® (product of Novo Nordisk)

The Results clearly show that the combination of the protease according to the invention with a conventional protease, cellulase or lipase a washing action strengthened.

Industrial applicability

The alkaline according to the invention Protease has excellent stability against a variety of surfactants Substances, resistance to fatty acids and high stability across from an oxidizing agent and is therefore useful as an enzyme for a detergent for one automatic dishwasher and as a laundry detergent, both containing a bleaching component.

SEQUENCE LISTING

Claims (9)

Alkaline protease, which: (a) one as in SEQ ID NO: 1 or 2 comprises amino acid sequence shown; or (B) the the amino acid sequence of (a) in which one or more amino acids are deleted, substituted or added are, and which has the following physico-chemical properties (I) active pH range: actively over a wide pH range of 4-13 and indicates at a pH from 6-12 an activity of 80% or more of the activity at the pH optimum; (ii) stable pH range: is stable over one pH range of 6-11 after treatment at 40 ° C for 30 minutes; (Iii) isoelectric point: has an isoelectric point of approximately 8.9-9.1 on; and (iv) Effect of a fatty acid: Casein-degrading activity is made by oleic acid not inhibited, and wherein the amino acid sequence is 90% or more is identical to SEQ ID NO. 1 or 2. An alkaline protease according to claim 1 which has an estimated molecular weight of about Has 43,000 when using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is determined. Nucleotide sequence comprising an alkaline protease according to claim 1 or 2 coded. Nucleotide sequence according to claim 3, which has a like under a sequence shown by SEQ ID Nos. 3 to 5. A vector comprising the nucleotide sequence of claim 3 or 4. A host cell comprising the nucleotide sequence of claim 3 or 4 or with the vector according to claim 5 is transformed. Microorganism, the FERM BP-6532, FERM BP-6533 or FERM BP-6534 is. Process for the preparation of alkaline protease according to claim 1, comprising breeding the host cell according to claim 6 or the microorganism according to claim 7 and collecting the protein from the culture medium. Detergent composition containing an alkaline Protease according to claim 1 or 2 or an alkaline protease as obtainable by the method according to claim 8th.